I-msoi homing endonuclease variants having novel substrate specificity and use thereof

ABSTRACT

An I-MsoI homing endonuclease variant able to cleave mutant I-MsoI sites having variation at positions ±8 to ±10, a vector encoding said variant, a cell, an animal or a plant modified by said vector. Use of said I-MsoI endonuclease variant and derived products for genetic engineering, genome therapy and antiviral therapy.

The invention relates also to an I-MsoI homing endonuclease varianthaving novel substrate specificity, to a vector encoding said variant,to a cell, an animal or a plant modified by said vector and to the useof said I-MsoI endonuclease variant and derived products for geneticengineering, genome therapy and antiviral therapy.

Among the strategies to engineer a given genetic locus, the use of rarecutting DNA endonucleases such as meganucleases has emerged as apowerful tool to increase homologous gene targeting through thegeneration of a DNA double strand break (DSB). Meganucleases recognizelarge (>12 bp) sequences, and can therefore cleave their cognate sitewithout affecting global genome integrity. Homing endonucleases, thenatural meganucleases, constitute several large families of proteinsencoded by mobile introns or inteins. Their target sequence is usuallyfound in homologous alleles that lack the intron or intein, and cleavageinitiates the transfer of the mobile element into the broken sequence bya mechanism of DSB-induced homologous recombination. I-SceI was thefirst homing endonuclease used to stimulate homologous recombinationover 1000-fold at a genomic target in mammalian cells (Choulika et al.,Mol. Cell. Biol., 1995, 15:1968-1973; Cohen-Tannoudji et al., Mol. Cell.Biol., 1998; 18:1444-1448; Donoho et al., Mol. Cell. Biol., 1998;18:4070-4078; Alwin et al., Mol. Ther., 2005, 12:610-617; Porteus, M.H., Mol. Ther., 2006, 13:438-446; Rouet et al., Mol. Cell. Biol., 1994,14:8096-8106). Recently, I-SceI was also used to stimulate targetedrecombination in mouse liver in vivo, and recombination could beobserved in up to 1% of hepatocytes (Gouble et al., J. Gene Med., 2006,8:616-622). However an inherent limitation of such a methodology is thatit requires the prior introduction of the natural cleavage site into thelocus of interest since the repertoire of sequences cleavable by naturalmeganucleases is too limited to address the complexity of the genomes,and there is usually no cleavable site in a chosen gene. To circumventthis limitation, significant efforts have been made over the past yearsto generate endonucleases with tailored cleavage specificities. Suchproteins could be used to cleave genuine chromosomal sequences and opennew perspectives for genome engineering in wide range of applications.For example, meganucleases could be used to induce the correction ofmutations linked with monogenic inherited diseases, and bypass the riskdue to the randomly inserted transgenes used in current gene therapyapproaches (Hacein-Bey-Abina et al., Science, 2003, 302, 415-419).

Fusion of Zinc-Finger Proteins (ZFPs) with the catalytic domain of theFokI, a class IIS restriction endonuclease, were used to make functionalsequence-specific endonucleases (Smith et al., Nucleic Acids Res., 1999,27, 674-681; Bibikova et al., Mol. Cell. Biol., 2001, 21, 289-297;Bibikova et al., Genetics, 2002, 161, 1169-1175; Bibikova et al.,Science, 2003, 300, 764; Porteus, M. H. and D. Baltimore, Science, 2003,300, 763-; Alwin et al., Mol. Ther., 2005, 12, 610-617; Urnov et al.,Nature, 2005, 435, 646-651; Porteus, M. H., Mol. Ther., 2006, 13,438-446). Such nucleases could recently be used for the engineering ofthe ILR2G gene in human cells from the lymphoid lineage (Urnov et al.,Nature, 2005, 435, 646-651).

The binding specificity of Cys2-His2 type Zinc-Finger Proteins, is easyto manipulate, probably because they represent a simple (specificitydriven by essentially four residues per finger), and modular system(Pabo et al., Annu. Rev. Biochem., 2001, 70, 313-340; Jamieson et al.,Nat. Rev. Drug Discov., 2003, 2, 361-368. Studies from the Pabo (Rebar,E. J. and C. O. Pabo, Science, 1994, 263, 671-673; Kim, J. S, and C. O.Pabo, Proc. Natl. Acad. Sci. USA, 1998, 95, 2812-2817), Klug (Choo, Y.and A. Klug, Proc. Natl. Acad. Sci. USA, 1994, 91, 11163-11167; IsalanM. and A. Klug, Nat. Biotechnol., 2001, 19, 656-660) and Barbas (Choo,Y. and A. Klug, Proc. Natl. Acad. Sci. USA, 1994, 91, 11163-11167;Isalan M. and A. Klug, Nat. Biotechnol., 2001, 19, 656-660) laboratoriesresulted in a large repertoire of novel artificial ZFPs, able to bindmost G/ANNG/ANNG/ANN sequences.

Nevertheless, ZFPs might have their limitations, especially forapplications requiring a very high level of specificity, such astherapeutic applications. The FokI nuclease activity in fusion acts as adimer, but it was recently shown that it could cleave DNA when only oneout of the two monomer was bound to DNA, or when the two monomers werebound to two distant DNA sequences (Catto et al., Nucleic Acids Res.,2006, 34, 1711-1720). Thus, specificity might be very degenerate, asillustrated by toxicity in mammalian cells (Porteus, M. H. and D.Baltimore, Science, 2003, 300, 763) and Drosophila (Bibikova et al.,Genetics, 2002, 161, 1169-1175; Bibikova et al., Science, 2003, 300,764-.).

Given their exquisite specificity, homing endonucleases may representideal scaffolds for engineering tailored endonucleases. Several studieshave shown that the DNA binding domain from LAGLIDADG proteins, the mostwidespread homing endonucleases (Chevalier, B. S, and Stoddard B. L.,Nucleic Acids Res. 2001; 29:3757-74) could be engineered. LAGLIDADGrefers to the only sequence actually conserved throughout the family andis found in one or more often two copies in the protein (Lucas et al.,Nucleic Acids Res., 2001, 29:960-969). Proteins with a single motif,such as I-CreI and I-MsoI, form homodimers and cleave palindromic orpseudo-palindromic DNA sequences, whereas the larger, double motifproteins, such as I-SceI are monomers and cleave non-palindromictargets. Several different LAGLIDADG proteins have been crystallized,and they exhibit a very striking conservation of the core structure thatcontrasts with the lack of similarity at the primary sequence level(Jurica et al., Mol. Cell., 1998; 2:469-476; Chevalier et al., Nat.Struct. Biol. 2001; 8:312-316; Chevalier et al., J. Mol. Biol., 2003,329:253-69, Moure et al., J. Mol. Biol., 2003, 334:685-695; Moure etal., Nat. Struct. Biol., 2002, 9:764-770; Ichiyanagi et al., J. Mol.Biol., 2000, 300:889-901; Duan et al., Cell, 1997, 89:555-564; Bolduc etal., Genes Dev., 2003, 17:2875-2888; Silva et al., J. Mol. Biol., 1999,286:1123-1136). In this core structure, two characteristic αββαββαfolds, contributed by two monomers, or by two domains in double LAGLIDAGproteins, are facing each other with a two-fold symmetry. DNA bindingdepends on the four β strands from each domain, folded into anantiparallel β-sheet, and forming a saddle on the DNA helix majorgroove. The catalytic core is central, with a contribution of bothsymmetric monomers/domains. In addition to this core structure, otherdomains can be found: for example, PI-SceI, an intein, has a proteinsplicing domain, and an additional DNA-binding domain (Moure et al.,Nat. Struct. Biol., 2002, 9:764-70, Grindl et al., Nucleic Acids Res.1998, 26:1857-1862).

Several LAGLIDAG proteins, including PI-SceI (Gimble et al., J. Mol.Biol., 2003, 334:993-1008), I-CreI (Seligman et al., Nucleic Acids Res.2002, 30:3870-3879; Sussman et al., J. Mol. Biol., 2004, 342:31-41;International PCT Applications WO 2006/097784, WO 2006/097853, WO2007/060495 and WO 2007/049156; Arnould et al., J. Mol. Biol., 2006,355, 443-458; Rosen et al., Nucleic Acids Res., 2006, 34, 4791-4800;Smith et al., Nucleic Acids Res., 2006, 34, e149), I-SceI (Doyon et al.,J Am Chem. Soc., 2006, 128:2477-2484) and I-MsoI (Ashworth et al.,Nature, 2006, 441:656-659) could be modified by rational orsemi-retional mutagenesis and screening to acquire new binding orcleavage specificities.

Another strategy was the creation of new meganucleases by domainswapping between I-CreI and I-DmoI, leading to the generation of ameganuclease cleaving the hybrid sequence corresponding to the fusion ofthe two half parent target sequences (Epinat et al., Nucleic Acids Res.,2003, 31:2952-2962; Chevalier et al., Mol. Cell. 2002, 10:895-905;International PCT Applications WO 03/078619 and WO 2004/031346).

Recently, semi rational design assisted by high throughput screeningmethods allowed to derive thousands of novel proteins from I-CreI (Smithet al., Nucleic Acids Res. 2006, 34, e149; Arnould et al., J. Mol.Biol., 2006, 355:443-458; International PCT Applications WO 2006/097784,WO 2006/097853, WO 2007/060495 and WO 2007/049156). In such an approach,a limited set of protein residues are chosen after examination ofprotein/DNA cocrystal structure, and randomized. Coupled withhigh-throughput screening (HTS) techniques, this method can rapidlyresult in the identification of hundreds of homing endonucleasesderivatives with modified specificities.

Furthermore, DNA-binding sub-domains that were independent enough toallow for a combinatorial assembly of mutations were identified (Smithet al., Nucleic Acids Res. 2006, 34, e 149; International PCTApplications WO 2007/049095 and WO 2007/057781). These findings allowedfor the production of a second generation of engineered I-CreIderivatives, cleaving chosen targets. This combinatorial strategy, hasbeen illustrated by the generation of meganucleases cleaving a naturalDNA target sequence located within the human RAG1 and XPC genes (Smithet al., Nucleic Acids Res., 2006, 34, e149; Arnould et Mol. Biol., 2007,371:49-65; International PCT Applications WO 2007/093836 and WO2007/093918).

However, although the capacity to combine up to four sub-domainsconsiderably increases the number of DNA sequences that can be targeted,it is still difficult to fully appreciate the range of sequences thatcan be reached. One of the most elusive factors is the impact of thefour central nucleotides of the I-CreI target site. Despite the absenceof base specific protein-DNA interactions in this region, in vitroselection of cleavable I-CreI targets from a library of randomlymutagenized sites revealed the importance of these 4 base-pairs forcleavage activity (Argast et al., J. Mol. Biol., 1998, 280:345-353.).More generally, it is unlikely that engineered meganucleases cleavingevery and any 22 by sequence could be derived from the sole I-CreIscaffold, and other proteins could be used as well, including monomericLAGLIDADG proteins.

I-MsoI is an homing endonuclease from Monomastix sp. It is a homodimericprotein and it shares 36% sequence identity with I-CreI. Its DNA targetis closely related to that of I-CreI, with only two differences atpositions −9 and +10 (FIG. 1). In addition, I-CreI and I-MsoI bothcleave each other's DNA target, and are therefore isoschizomers(Chevalier et al., J. Mol. Biol. 2003, 329:253-69). The structure ofI-MsoI in complex with its DNA target has been solved (Chevalier et al.,J. Mol. Biol., 2003, 329:253-269) and is shown in FIG. 2. Structureanalysis showed that in spite of DNA target similarity, DNA recognitionby I-MsoI and I-CreI depend on a different sets of interaction patterns.

A single I-MsoI variants (K28L, T83R) with novel cleavage specificityfor positions ±6 was designed by using a pure rational process, relyingon a computational approach (Ashworth et al., Nature, 2006,441:656-659).

Computational models were used to identify specific amino acid residuesthat specifically interact with the I-MsoI site and predict specificamino acid substitutions which alter the specificity towards individualbases within the I-MsoI site sequence (International PCT Application WO2007/047859). According to these predictions, the specificity towardsthe nucleotides at positions ±8, ±9 and ±10 of the I-MsoI site might bechanged by specific substitutions of I30, S43 and I85 (position ±8), Q41and R32 (position ±9), and Y35 and R32 (position ±10), respectively(Table 2 page 41 of WO 2007/047859). However, this approach was notvalidated experimentally and no I-MsoI variant having the predictedmutations was shown to have indeed a modified cleavage specificitytowards the nucleotides at positions ±8, ±9 and ±10 of the I-MsoI site.

By using a semi-rational approach very similar to the one previouslydescribed to engineer the I-CreI protein, the inventors have engineeredaround one hundred of novel I-MsoI variants which, altogether, target 31mutant DNA target sites differing at positions ±10, ±9, and ±8.

These variants have mutations at position 32 and/or 41 of I-MsoIsequence which are different to those predicted in the International PCTApplication WO 2007/047859. Furthermore, the inventors have demonstratedthat contrary to what is stated in Table 2 of WO 2007/047859, there isno correlation between a specific amino acid residue at position 32 and41 and a particular nucleotide g, t, a or c at position ±10, ±9, and ±8.These results indicate that although, the structure of 1-MsoI in complexwith its DNA target has been solved, changing the specificity of MsoI isa complex problem.

These variants having new substrate specificity towards nucleotides ±8,±9, and/or ±10, increase the number of DNA sequences that can betargeted with meganucleases. Potential applications include geneticengineering, genome engineering, gene therapy and antiviral therapy.

Thus, the invention concerns a method for engineering a I-MsoI homingendonuclease variant having novel substrate specificity, comprising:

(a) constructing a library of I-MsoI variants having amino acidvariation at one or more positions of I-MsoI amino acid sequenceselected from the group consisting of: P31, R32, P33, Y35, Q41 and S43,and

(b) assaying the cleavage activity of the variants from step (a) towardsa panel of DNA targets consisting of mutant I-MsoI sites wherein one ormore nucleotides at positions ±8 to 10 have been replaced with differentnucleotides, and

(c) selecting/screening the variants from step (b) having a pattern ofcleaved DNA targets that is different from that of the parent I-MsoIhoming endonuclease.

DEFINITIONS

-   -   Amino acid residues in a polypeptide sequence are designated        herein according to the one-letter code, in which, for example,        P means Pro or Proline residue, R means Arg or Arginine residue        and Y means Tyr or Tyrosine residue.    -   Nucleotides are designated as follows: one-letter code is used        for designating the base of a nucleoside: a is adenine, t is        thymine, c is cytosine, and g is guanine. For the degenerated        nucleotides, r represents g or a (purine nucleotides), k        represents g or t, s represents g or c, w represents a or t, m        represents a or c, y represents t or c (pyrimidine nucleotides),        d represents g, a or t, v represents g, a or c, b represents g,        t or c, h represents a, t or c, and n represents g, a, t or c.    -   by “meganuclease”, is intended an endonuclease having a        double-stranded DNA target sequence of 12 to 45 bp.    -   by “homodimeric LAGLIDADG homing endonuclease” is intended a        wild-type homodimeric LAGLIDADG homing endonuclease having a        single LAGLIDADG motif and cleaving palindromic DNA target        sequences, such as I-CreI or I-MsoI or a functional variant        thereof.

by “I-MsoI” is intended the wild-type I-MsoI having the sequence pdbaccession code 1M5X_A or 1M5X_B (SEQ ID NO: 1).

-   -   by “I-MsoI homing endonuclease variant”, “meganuclease variant”        or “variant” is intended a protein obtained by replacing at        least one amino acid of 1-MsoI sequence with a different amino        acid. According to the invention, the amino acid residue which        is mutated is indicated by its position in I-MsoI sequence SEQ        ID NO: 1. For example, P31 refers to the proline residue at        position 31 of the sequence SEQ ID NO: 1.    -   by “functional variant” is intended a I-MsoI homing endonuclease        variant which is able to cleave a DNA target, preferably a new        DNA target which is not cleaved by I-MsoI. For example, such        variants have amino acid variation at positions interacting        directly or indirectly with the DNA target sequence.    -   by “parent I-MsoI homing endonuclease” is intended I-MsoI or a        functional variant thereof. Said parent I-MsoI homing        endonuclease is a dimer (homodimer or heterodimer) comprising        two I-MsoI homing endonuclease monomers/core domains which are        associated in a functional endonuclease able to cleave a        double-stranded DNA target of 22 to 24 bp.    -   by “homing endonuclease variant with novel specificity” is        intended a variant having a pattern of cleaved DNA targets        (cleavage profile) different from that of the parent homing        endonuclease. The variants may cleave less targets (restricted        profile) or more targets than the parent homing endonuclease.        Preferably, the variant is able to cleave at least one target        that is not cleaved by the parent homing endonuclease.

The terms “novel specificity”, “modified specificity”, “alteredspecificity”, “novel cleavage specificity”, “novel substratespecificity” which are equivalent and used indifferently, refer to thespecificity of the variant towards the nucleotides of the DNA targetsequence.

-   -   by “homing endonuclease domain”, “domain” or “core domain” is        intended the “LAGLIDADG homing endonuclease core domain” which        is the characteristic α₁β₁β₂α₂β₃β₄α₃ fold of the homing        endonucleases of the LAGLIDADG family corresponding to a        sequence of about one hundred amino acid residues. Said domain        comprises four beta-strands (β₁, β₂, β₃, β₄) folded in an        antiparallel beta-sheet which interacts with one half of the DNA        target of a homing endonuclease and is able to associate with        the other domain of the same homing endonuclease which interacts        with the other half of the DNA target to form a functional        endonuclease able to cleave said DNA target. For example, in the        case of the dimeric homing endonuclease I-MsoI (170 amino        acids), the LAGLIDADG homing endonuclease core domain        corresponds to the residues 9 to 97.    -   by “subdomain” is intended the region of a LAGLIDADG homing        endonuclease core domain which interacts with a distinct part of        a homing endonuclease DNA target half-site. Two different        subdomains behave independently and the mutation in one        subdomain does not alter the binding and cleavage properties of        the other subdomain. Therefore, two subdomains bind distinct        part of a homing endonuclease DNA target half-site.    -   by “beta-hairpin” is intended two consecutive beta-strands of        the antiparallel beta-sheet of a LAGLIDADG homing endonuclease        core domain ((β₁β₂ or, β₃β₄) which are connected by a loop or a        turn,    -   by “single-chain meganuclease”, “single-chain chimeric        meganuclease”, “single-chain meganuclease derivative”,        “single-chain chimeric meganuclease derivative” or “single-chain        derivative” is intended a meganuclease comprising two LAGLIDADG        homing endonuclease domains or core domains linked by a peptidic        spacer. The single-chain meganuclease is able to cleave a        chimeric DNA target sequence comprising one different half of        each parent meganuclease target sequence.    -   by “DNA target”, “DNA target sequence”, “target sequence”,        “target-site”, “target”, “site”; “site of interest”;        “recognition site”, “recognition sequence”, “homing recognition        site”, “homing site”, “cleavage site” is intended a 20 to 24 by        double-stranded palindromic, partially palindromic        (pseudo-palindromic) or non-palindromic polynucleotide sequence        that is recognized and cleaved by a LAGLIDADG homing        endonuclease such as I-MsoI, or a variant, or a single-chain        chimeric meganuclease derived from I-MsoI. These terms refer to        a distinct DNA location, preferably a genomic location, at which        a double stranded break (cleavage) is to be induced by the        meganuclease. The DNA target is defined by the 5′ to 3′ sequence        of one strand of the double-stranded polynucleotide. Cleavage of        the DNA target occurs at the nucleotides at positions +2 and −2,        respectively for the sense and the antisense strand. Unless        otherwise indicated, the position at which cleavage of the DNA        target by an I-MsoI meganuclease variant occurs, corresponds to        the cleavage site on the sense strand of the DNA target.    -   by “I-MsoI site” is intended a 22 to 24 by double-stranded DNA        sequence which is cleaved by I-MsoI. I-MsoI sites include the        wild-type (natural) non-palindromic I-MsoI homing site (SEQ ID        NO: 2; FIG. 1), the I-CreI homing site (SEQ ID NO: 3) and the        derived palindromic sequences which are presented in FIG. 1,        such as the sequence        5′-c⁻¹¹a⁻¹⁰a⁻⁹a⁻⁸a⁻⁷c⁻⁶g⁻⁵t⁻⁴c⁻³g⁻²t⁻¹a₊₁c₊₂g₊₃a₊₄c₊₅g₊₆t₊₇t₊₈t₉t₊₁₀g₊₁₁        also called C1221 (SEQ ID NO: 4).    -   by “DNA target half-site”, “half cleavage site” or half-site” is        intended the portion of the DNA target which is bound by each        LAGLIDADG homing endonuclease core domain.    -   by “chimeric DNA target” or “hybrid DNA target” is intended the        fusion of a different half of two parent meganuclease target        sequences. In addition at least one half of said target may        comprise the combination of nucleotides which are bound by at        least two separate subdomains (combined DNA target).    -   by “vector” is intended a nucleic acid molecule capable of        transporting another nucleic acid to which it has been linked.    -   by “homologous” is intended a sequence with enough identity to        another one to lead to a homologous recombination between        sequences, more particularly having at least 95% identity,        preferably 97% identity and more preferably 99%.    -   “Identity” refers to sequence identity between two nucleic acid        molecules or polypeptides. Identity can be determined by        comparing a position in each sequence which may be aligned for        purposes of comparison. When a position in the compared sequence        is occupied by the same base, then the molecules are identical        at that position. A degree of similarity or identity between        nucleic acid or amino acid sequences is a function of the number        of identical or matching nucleotides at positions shared by the        nucleic acid sequences. Various alignment algorithms and/or        programs may be used to calculate the identity between two        sequences, including FASTA, or BLAST which are available as a        part of the GCG sequence analysis package (University of        Wisconsin, Madison, Wis.), and can be used with, e.g., default        settings.    -   “individual” includes mammals, as well as other vertebrates        (e.g., birds, fish and reptiles). The terms “mammal” and        “mammalian”, as used herein, refer to any vertebrate animal,        including monotremes, marsupials and placental, that suckle        their young and either give birth to living young (eutharian or        placental mammals) or are egg-laying (metatharian or        nonplacental mammals). Examples of mammalian species include        humans and other primates (e.g., monkeys, chimpanzees), rodents        (e.g., rats, mice, guinea pigs) and others such as for example:        cows, pigs and horses.    -   “genetic disease” refers to any disease, partially or        completely, directly or indirectly, due to an abnormality in one        or several genes. Said abnormality can be a mutation, an        insertion or a deletion. Said mutation can be a punctual        mutation. Said abnormality can affect the coding sequence of the        gene or its regulatory sequence. Said abnormality can affect the        structure of the genomic sequence or the structure or stability        of the encoded mRNA. Said genetic disease can be recessive or        dominant. Such genetic disease could be, but are not limited to,        cystic fibrosis, Huntington's chorea, familial        hyperchoiesterolemia (LDL receptor defect), hepatoblastoma,        Wilson's disease, congenital hepatic porphyrias, inherited        disorders of hepatic metabolism, Lesch Nyhan syndrome, sickle        cell anemia, thalassaemias, xeroderma pigmentosum, Fanconi's        anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's        syndrome, retinoblastoma, Duchenne's muscular dystrophy, and        Tay-Sachs disease.    -   by mutation is intended the substitution, deletion, insertion of        one or more nucleotides/amino acids in a polynucleotide (cDNA,        gene) or a polypeptide sequence. Said mutation can affect the        coding sequence of a gene or its regulatory sequence. It may        also affect the structure of the genomic sequence or the        structure/stability of the encoded mRNA.

According, to an advantageous embodiment of said method, the library instep a) comprises the replacement of the initial amino acid(s) with S,P, T, A, Y, H, Q, N, K, D, E, C, W, R and G.

The library in step (a) is prepared according to standard methods whichare well-known in the art. For example, the library may be produced byamplifying fragments overlapping in the region of the mutation(s) withdegenerated primer(s) to allow degeneracy at the position(s) of themutation(s).

According to an advantageous embodiment of said method, the library instep (a) is a combinatorial library having diversity at two or threepositions of I-MsoI sequence. For example, the library has diversity atpositions 32 and 41, 32 and 43, 32 and 35, 32, 41 and 43, or 31, 32 and33. Combinatorial libraries may be generated as described inInternational PCT Applications WO 2004/067736, WO 2006/097853, WO2007/057781 and WO 2007/049156; Arnould et al., J. Mol. Biol., 2006,355, 443-458; Smith et al., Nucleic Acids Res., 2006, 34, e149.

The parent I-MsoI homing endonuclease (initial scaffold protein) whichis used for preparing the library of variants may be I-MsoI, for examplethe sequence SEQ ID NO: 1 or a functional variant of I-MsoI variant asdefined above. In addition, one or more residues may be inserted at theNH₂ terminus and/or COOH terminus of the scaffold protein. Additionalcodons may be added at the 5′ or 3′ end of the I-MsoI coding sequence tointroduce restrictions sites which are used for cloning into variousvectors. An example of said sequence is SEQ ID NO: 105 which has analanine (A) residue inserted after the first methionine residue and analanine and an aspartic acid (AD) residues inserted after the C-terminalproline residue. These sequences allow having DNA coding sequencescomprising the NcoI (ccatgg) and EagI (cggccg) restriction sites whichare used for cloning into various vectors. A tag (epitope orpolyhistidine sequence) may also be introduced at the NH₂ terminusand/or COOH terminus; said tag is useful for the detection and/or thepurification of the meganuclease.

According to the method of the invention, the library of variants fromstep (a) may comprise additional mutations in order to improve thebinding and/or cleavage activity of the mutants towards the DNAtarget(s) of interest. Said mutations may be at other positions indirect or indirect (via a water molecule) interaction with the phosphatebackbone or with the nucleotide bases of the DNA target. Furthermore,random mutations may also be introduced on the whole variant or in partof the variant, in order to improve the binding and/or cleavage activityof the variant towards the DNA target(s) of interest. This may beperformed by generating random mutagenesis libraries on a pool ofvariants, according to standard mutagenesis methods which are well-knownin the art and commercially available. The additional mutations (randomor site-specific) and the mutation(s) of P31, R32, P33, Y35, Q41 and/orS43 may be introduced simultaneously or subsequently.

According to the method of the invention, the DNA target in step b) maybe palindromic, non-palindromic or pseudo-palindromic. Preferably, theDNA target in step b) is a palindromic target comprising the sequence:c⁻¹¹n⁻¹⁰n⁻⁹n⁻⁸a⁻⁷c⁻⁶g⁻⁵t⁻⁴c⁻³g⁻²t⁻¹a₊₁c₊₂g₊₃a₊₄c₊₅g₊₆t₊₇n₊₈n₊₉n₊₁₀g₊₁₁,wherein n is a, t, c, or g (SEQ ID NO: 5); this target derives fromC1221 (SEQ ID NO: 4, FIG. 1).

According to the method of the invention, step (b) may be performed byusing a cleavage assay in vitro or in vivo, as described in theInternational PCT Application WO 2004/067736. Preferably, step (b) isperformed in vivo, under conditions where the double-strand break in themutated DNA target sequence which is generated by said variant leads tothe activation of a positive selection marker or a reporter gene, or theinactivation of a negative selection marker or a reporter gene, byrecombination-mediated repair of said DNA double-strand break. Forexample, the cleavage activity of the I-MsoI variant of the inventionmay be measured by a direct repeat recombination assay, in yeast ormammalian cells, using a reporter vector, as described in the PCTApplication WO 2004/067736. The reporter vector comprises two truncated,non-functional copies of a reporter gene (direct repeats) and a DNAtarget sequence within the intervening sequence, cloned in a yeast or amammalian expression vector. The DNA target sequence is palindromic andderived from a I-MsoI site such as C1221, by substitution of one tothree nucleotides at positions ±8 to 10 (FIG. 1). Expression of afunctional I-MsoI variant which is able to cleave the DNA targetsequence, induces homologous recombination between the direct repeats,resulting in a functional reporter gene, whose expression can bemonitored by appropriate assay.

According to another advantageous embodiment of said method, step (c)comprises the selection of variants able to cleave at least one DNAtarget that is not cleaved by I-MsoI. The 18 targets which are cleavedby I-MsoI are presented in FIGS. 7 and 8.

According to another advantageous embodiment of said method, itcomprises a further step d₁) of expressing one variant obtained in stepc), so as to allow the formation of homodimers.

According to another advantageous embodiment of said method, itcomprises a further step d₂) of co-expressing one variant obtained instep c) and I-MsoI or a functional variant thereof, so as to allow theformation of heterodimers. Preferably, two different variants obtainedin step c) are co-expressed.

For example, host cells may be modified by one or two recombinantexpression vector(s) encoding said variant(s). The cells are thencultured under conditions allowing the expression of the variant(s) andthe homodimers/heterodimers which are formed are then recovered from thecell culture.

According to the method of the invention, single-chain chimericmeganucleases may be constructed by the fusion of one monomer/domainvariant obtained in step (c) with a homing endonuclease domain/monomer.Said monomer/domain from a wild-type LAGLIDADG homing endonuclease or afunctional variant thereof. Preferably, the two domain(s)/monomer(s) areconnected by a peptidic linker. More preferably, the single-chainmeganuclease comprises two monomers, each from a different variantobtained in step (c); said single-chain meganuclease is able cleave anon-palindromic chimeric target comprising one different half of eachvariant DNA target.

Methods for constructing single-chain chimeric meganucleases derivedfrom homing endonucleases are well-known in the art (Epinat et al.,Nucleic Acids Res., 2003, 31, 2952-62; Chevalier et al., Mol. Cell.,2002, 10, 895-905; Steuer et al., Chembiochem., 2004, 5, 206-13;International PCT Applications WO 03/078619 and WO 2004/031346). Any ofsuch methods, may be applied for constructing single-chain chimericmeganucleases derived from the variants as defined in the presentinvention.

The subject matter of the present invention is also a I-MsoI homingendonuclease variant obtainable by the method as defined above, saidvariant having at least one mutation at position 31, 32, 33, 35, 41,and/or 43 of I-MsoI, and a cleavage pattern towards a panel of mutantI-MsoI sites having variation at positions ±8 to 10, that is differentfrom that of I-MsoI.

According to an advantageous embodiment of said I-MsoI variant, itcomprises at least the replacement of Q41 with N, G, Y, R, T, S, P, C,H, K, A or W. Preferably Q41 is replaced with N, G, Y, T, S, P, C, H, Aor W.

According to another advantageous embodiment of said I-MsoI variant, itcomprises at least the replacement of R32 with K, Q, A, H, S, G, D, W,P, T, C, E and N. Preferably R32 is replaced with Q, A, H, S, G, D, W,P, T, C, and N.

According to another advantageous embodiment of said I-MsoI variant, itcomprises at least the replacement of P31 or P33 with S, T, A, Y, H, Q,N, K, D, E, C, W, R or G.

According to another advantageous embodiment of said I-MsoI variant, itcomprises at least the replacement of Y35 with S, P, T, A, H, Q, N, D,E, C, W, or G.

According to another advantageous embodiment of said I-MsoI variant, itcomprises at least the replacement of S43 with P, T, A, Y, H, N, D, C,W, or G.

According to another advantageous embodiment of said I-MsoI variant, itcomprises at least one additional mutation at a position of I-MsoI thatimproves the binding and/or the cleavage activity towards the DNAtarget, said position being selected from the group consisting of: T3,K4, T6, L7, K36, D37, K39, Y40, V42, F48, F55, Y82, T88, I93, L97, N109,I134, A145, T151 and A163. Preferably, said mutation is selected fromthe group consisting of: T3A, K4M, T6A, L7S, K36N, K36I, D37N, K39N,K39R, K39T, Y40S, V42M, F48Y, F55V, F55I, Y82H, T88A, I93M, L97S, N109S,I134V, I134M, A145V, T151A and A163V.

The invention includes a first series of I-MsoI variants able to cleaveat least one DNA target having variation at positions ±8 to 10, that isnot cleaved by I-MsoI, said variants comprising mutations selected fromthe group consisting of: R32K and Q41N; Q41T; R32S and Q41S; R32A andQ41R; R32W and Q41N; R32S and Q41R; R32Q and Q41R; Q41Y; Q41N; Q41C;R32T and Q41R; Q41H; R32W and Q41T; Q41S; Q41G; R32E and Q41T; R32Q andQ41A; R32G and Q41Y; Q41P; R32P and Q41T; Q41A; T3A, R32Q and Q41P; Q41Nand T88A; R32S and Q41N; R32Q, Q41P and F48Y; R32S, K39N and Q41S; R32D,Q41K and L97S; R32H, Q41K and A145V; P33S and Q41C; Y35F and Q41K; R32C,K39T and Q41K; R32A and Q41P; R32T, Y40S and Q41S; R32G and Q41R; R32Hand Q41P; R32E, K36E and Q41T, R32P and Q41P. Examples of said variantsare the sequences SEQ ID NO: 6 to 42 (FIG. 8).

Preferably, said DNA target that is not cleaved by I-MsoI comprises anucleotide triplet at positions −10 to −8, which is selected from thegroup consisting of: aag, gtg, gta, gtt, gcc, tga, taa, cac, cta, tca,cca, ccc and cgc and/or a nucleotide triplet at positions ±8 to +10,which is the reverse complementary sequence of said nucleotide tripletat positions −10 to −8.

The invention includes also a second series of I-MsoI variants having acleavage pattern towards targets having variation at positions ±8 to 10which is more restricted than that of I-MsoI, said variants comprisingmutations selected from the group consisting of: R32Q and Q41G; R32A andQ41Y; R32H and Q41R; R32D and Q41P; R32D and Q41R; R32Q and Q41N; R32Pand Q41R; R32K and Q41Y; R32K and Q41T; R32K and Q41H; R32K, Q41G andV42M; R32S and Q41Y; R32H and Q41G; R32H and Q41H; R32Q and Q41S; R32Sand Q41K; R32A and Q41S; R32H and Q41S; R32C and Q41H; R32H and Q41N;R32C and Q41T; R32S and Q41H; R32T and Q41K; R32A and Q41H; R32G andQ41K; R32S and Q41P; R32H and Q41T; R32Q and Q41H; R32Q and Q41T; R32Kand Q41R; R32E and Q41W; R32K and Q41S; R32N and Q41N; R32H and Q41C;R32S and Q41A; Q41K and F55I; T6A, Q41K and 193M; R32E, Q41T and N109S;R32G and Q41W; K4M, R32T and Q41R; Y35S and D37N; R32H and Q41A; K39Rand Q41S; L7S, R32K and Q41H; K36N and Q41N; P33L and Q41P; R32T, Q41Rand T151A; Q41Y and A163V; R32S, Q41H and I134V; Q41T and Y82H; R32H,D37N and Q41T; Q41N and P43N; R32K, Q41S and I134M; R32A, Q41K and F55V;Q41S and F48Y. Examples of said variants are the sequences SEQ ID NO:43, 44, 46 to 65 and 67 to 99 (FIG. 8).

The I-MsoI variant of the invention may be an homodimer or anheterodimer.

According to another advantageous embodiment of said I-MsoI variant, itis an heterodimer comprising monomers from two different variants.

The subject-matter of the present invention is also a single-chainchimeric meganuclease (fusion protein) derived from an I-MsoI variant asdefined above. The single-chain meganuclease may comprise two I-MsoImonomers, two I-MsoI core domains or a combination of both. Preferably,the two monomers/core domains or the combination of both, are connectedby a peptidic linker.

The meganuclease of the invention includes both the meganuclease variantand the single-chain meganuclease derivative.

The subject-matter of the present invention is also a polynucleotidefragment encoding a variant or a single-chain chimeric meganuclease asdefined above; said polynucleotide may encode one monomer of anhomodimeric or heterodimeric variant, or two domains/monomers of asingle-chain chimeric meganuclease.

The subject-matter of the present invention is also a recombinant vectorfor the expression of a variant or a single-chain meganuclease accordingto the invention. The recombinant vector comprises at least onepolynucleotide fragment encoding a variant or a single-chainmeganuclease, as defined above. In a preferred embodiment, said vectorcomprises two different polynucleotide fragments, each encoding one ofthe monomers of an heterodimeric variant.

A vector which can be used in the present invention includes, but is notlimited to, a viral vector, a plasmid, a RNA vector or a linear orcircular DNA or RNA molecule which may consists of a chromosomal, nonchromosomal, semi-synthetic or synthetic nucleic acids. Preferredvectors are those capable of autonomous replication (episomal vector)and/or expression of nucleic acids to which they are linked (expressionvectors). Large numbers of suitable vectors are known to those of skillin the art and commercially available.

Viral vectors include retrovirus, adenovirus, parvovirus (e.g.adeno-associated viruses), coronavirus, negative strand RNA viruses suchas orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies andvesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai),positive strand RNA viruses such as picornavirus and alphavirus, anddouble-stranded DNA viruses including adenovirus, herpesvirus (e.g.,Herpes Simplex virus types 1 and 2, Epstein-Barr virus,cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox).Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses,papovavirus, hepadnavirus, and hepatitis virus, for example. Examples ofretroviruses include: avian leukosis-sarcoma, mammalian C-type, B-typeviruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin,J. M., Retroviridae: The viruses and their replication, In FundamentalVirology, Third Edition, B. N. Fields, et al., Eds., Lippincott-RavenPublishers, Philadelphia, 1996).

Preferred vectors include lentiviral vectors, and particularly selfinactivating lentiviral vectors.

Vectors can comprise selectable markers, for example: neomycinphosphotransferase, histidinol dehydrogenase, dihydrofolate reductase,hygromycin phosphotransferase, herpes simplex virus thymidine kinase,adenosine deaminase, glutamine synthetase, and hypoxanthine-guaninephosphoribosyl transferase for eukaryotic cell culture; TRP1, URA3 andLEU2 for S. cerevisiae; tetracycline, rifampicin or ampicillinresistance in E. coli.

Preferably said vectors are expression vectors, wherein the sequence(s)encoding the variant/single-chain meganuclease of the invention isplaced under control of appropriate transcriptional and translationalcontrol elements to permit production or synthesis of said variant.Therefore, said polynucleotide is comprised in an expression cassette.More particularly, the vector comprises a replication origin, a promoteroperatively linked to said encoding polynucleotide, a ribosome-bindingsite, an RNA-splicing site (when genomic DNA is used), a polyadenylationsite and a transcription termination site. It also can comprise anenhancer. Selection of the promoter will depend upon the cell in whichthe polypeptide is expressed. Preferably, when said variant is anheterodimer, the two polynucleotides encoding each of the monomers areincluded in one vector which is able to drive the expression of bothpolynucleotides, simultaneously. Suitable promoters include tissuespecific and/or inducible promoters. Examples of inducible promotersare: eukaryotic metallothionine promoter which is induced by increasedlevels of heavy metals, prokaryotic lacZ promoter which is induced inresponse to isopropyl-β-D-thiogalacto-pyranoside (IPTG) and eukaryoticheat shock promoter which is induced by increased temperature. Examplesof tissue specific promoters are skeletal muscle creatine kinase,prostate-specific antigen (PSA), α-antitrypsin protease, humansurfactant (SP) A and B proteins, β-casein and acidic whey proteingenes.

According to another advantageous embodiment of said vector, it includesa targeting construct comprising sequences sharing homologies with theregion surrounding the genomic DNA cleavage site as defined above.

Alternatively, the vector coding for an I-MsoI variant/single-chainmeganuclease and the vector comprising the targeting construct aredifferent vectors.

More preferably, the targeting DNA construct comprises:

a) sequences sharing homologies with the region surrounding the genomicDNA cleavage site as defined above, and

b) a sequence to be introduced flanked by sequences as in a).

Preferably, homologous sequences of at least 50 bp, preferably more than100 by and more preferably more than 200 bp are used. Therefore, thetargeting DNA construct is preferably from 200 pb to 6000 pb, morepreferably from 1000 pb to 2000 pb. Indeed, shared DNA homologies arelocated in regions flanking upstream and downstream the site of thebreak and the DNA sequence to be introduced should be located betweenthe two arms. The sequence to be introduced is preferably a sequencewhich repairs a mutation in the gene of interest (gene correction orrecovery of a functional gene), for the purpose of genome therapy.Alternatively, it can be any other sequence used to alter thechromosomal DNA in some specific way including a sequence used to modifya specific sequence, to attenuate or activate the gene of interest, toinactivate or delete the gene of interest or part thereof, to introducea mutation into a site of interest or to introduce an exogenous gene orpart thereof. Such chromosomal DNA alterations are used for genomeengineering (animal models/human recombinant cell lines).

The invention also concerns a prokaryotic or eukaryotic host cell whichis modified by a polynucleotide or a vector as defined above, preferablyan expression vector.

The invention also concerns a non-human transgenic animal or atransgenic plant, characterized in that all or part of their cells aremodified by a polynucleotide or a vector as defined above.

As used herein, a cell refers to a prokaryotic cell, such as a bacterialcell, or eukaryotic cell, such as an animal, plant or yeast cell.

The subject-matter of the present invention is further the use of ameganuclease, one or two derived polynucleotide(s), preferably includedin expression vector(s), a cell, a transgenic plant, a non-humantransgenic mammal, as defined above, for molecular biology, for in vivoor in vitro genetic engineering, and for in vivo or in vitro genomeengineering, for non-therapeutic purposes.

Molecular biology includes with no limitations, DNA restriction and DNAmapping. Genetic and genome engineering for non therapeutic purposesinclude for example (i) gene targeting of specific loci in cellpackaging lines for protein production, (ii) gene targeting of specificloci in crop plants, for strain improvements and metabolic engineering,(iii) targeted recombination for the removal of markers in geneticallymodified crop plants, (iv) targeted recombination for the removal ofmarkers in genetically modified microorganism strains (for antibioticproduction for example).

According to an advantageous embodiment of said use, it is for inducinga double-strand break in a site of interest comprising a DNA targetsequence, thereby inducing a DNA recombination event, a DNA loss or celldeath.

According to the invention, said double-strand break is for: repairing aspecific sequence, modifying a specific sequence, restoring a functionalgene in place of a mutated one, attenuating or activating an endogenousgene of interest, introducing a mutation into a site of interest,introducing an exogenous gene or a part thereof, inactivating ordetecting an endogenous gene or a part thereof, translocating achromosomal arm, or leaving the DNA unrepaired and degraded.

The subject-matter of the present invention is also a method of geneticengineering, characterized in that it comprises a step of double-strandnucleic acid breaking in a site of interest located on a vectorcomprising a DNA target as defined hereabove, by contacting said vectorwith a meganuclease as defined above, thereby inducing an homologousrecombination with another vector presenting homology with the sequencesurrounding the cleavage site of said meganuclease.

The subjet-matter of the present invention is also a method of genomeengineering, characterized in that it comprises the following steps: 1)double-strand breaking a genomic locus comprising at least one DNAtarget of a meganuclease as defined above, by contacting said targetwith said meganuclease; 2) maintaining said broken genomic locus underconditions appropriate for homologous recombination with a targeting DNAconstruct comprising the sequence to be introduced in said locus,flanked by sequences sharing homologies with the targeted locus.

The subject-matter of the present invention is also a method of genomeengineering, characterized in that it comprises the following steps: 1)double-strand breaking a genomic locus comprising at least one DNAtarget of a meganuclease as defined above, by contacting said cleavagesite with said meganuclease; 2) maintaining said broken genomic locusunder conditions appropriate for homologous recombination withchromosomal DNA sharing homologies to regions surrounding the cleavagesite.

The subject-matter of the present invention is also the use of at leastone meganuclease as defined above, one or two derived polynucleotide(s),preferably included in expression vector(s), as defined above, for thepreparation of a medicament for preventing, improving or curing agenetic disease in an individual in need thereof, said medicament beingadministrated by any means to said individual.

The subject-matter of the present invention is also a method forpreventing, improving or curing a genetic disease in an individual inneed thereof, said method comprising the step of administering to saidindividual a composition comprising at least a meganuclease as definedabove, by any means.

In this case, the use of the meganuclease as defined above, comprises atleast the step of (a) inducing in somatic tissue(s) of the individual adouble stranded cleavage at a site of interest of a gene comprising atleast one recognition and cleavage site of said meganuclease, and (b)introducing into the individual a targeting DNA, wherein said targetingDNA comprises (1) DNA sharing homologies to the region surrounding thecleavage site and (2) DNA which repairs the site of interest uponrecombination between the targeting DNA and the chromosomal DNA. Thetargeting DNA is introduced into the individual under conditionsappropriate for introduction of the targeting DNA into the site ofinterest.

According to the present invention, said double-stranded cleavage isinduced, either in toto by administration of said meganuclease to anindividual, or ex vivo by introduction of said meganuclease into somaticcells removed from an individual and returned into the individual aftermodification.

In a preferred embodiment of said use, the meganuclease is combined witha targeting DNA construct comprising a sequence which repairs a mutationin the gene flanked by sequences sharing homologies with the regions ofthe gene surrounding the genomic DNA cleavage site of said meganuclease,as defined above. The sequence which repairs the mutation is either afragment of the gene with the correct sequence or an exon knock-inconstruct.

For correcting a gene, cleavage of the gene occurs in the vicinity ofthe mutation, preferably, within 500 bp of the mutation. The targetingconstruct comprises a gene fragment which has at least 200 by ofhomologous sequence flanking the genomic DNA cleavage site (minimalrepair matrix) for repairing the cleavage, and includes the correctsequence of the gene for repairing the mutation. Consequently, thetargeting construct for gene correction comprises or consists of theminimal repair matrix; it is preferably from 200 pb to 6000 pb, morepreferably from 1000 pb to 2000 pb.

For restoring a functional gene, cleavage of the gene occurs upstream ofa mutation. Preferably said mutation is the first known mutation in thesequence of the gene, so that all the downstream mutations of the genecan be corrected simultaneously. The targeting construct comprises theexons downstream of the genomic DNA cleavage site fused in frame (as inthe cDNA) and with a polyadenylation site to stop transcription in 3′.The sequence to be introduced (exon knock-in construct) is flanked byintrons or exons sequences surrounding the cleavage site, so as to allowthe transcription of the engineered gene (exon knock-in gene) into amRNA able to code for a functional protein. For example, the exonknock-in construct is flanked by sequences upstream and downstream.

The subject-matter of the present invention is also the use of at leastone meganuclease as defined above, one or two derived polynucleotide(s),preferably included in expression vector(s), as defined above for thepreparation of a medicament for preventing, improving or curing adisease caused by an infectious agent that presents a DNA intermediate,in an individual in need thereof, said medicament being administrated byany means to said individual.

The subject-matter of the present invention is also a method forpreventing, improving or curing a disease caused by an infectious agentthat presents a DNA intermediate, in an individual in need thereof, saidmethod comprising at least the step of administering to said individuala composition as defined above, by any means.

The subject-matter of the present invention is also the use of at leastone meganuclease as defined above, one or two polynucleotide(s),preferably included in expression vector(s), as defined above, in vitro,for inhibiting the propagation, inactivating or deleting an infectiousagent that presents a DNA intermediate, in biological derived productsor products intended for biological uses or for disinfecting an object.

The subject-matter of the present invention is also a method fordecontaminating a product or a material from an infectious agent thatpresents a DNA intermediate, said method comprising at least the step ofcontacting a biological derived product, a product intended forbiological use or an object, with a composition as defined above, for atime sufficient to inhibit the propagation, inactivate or delete saidinfectious agent.

In a particular embodiment, said infectious agent is a virus. Forexample said virus is an adenovirus (Ad11, Ad21), herpesvirus (HSV, VZV,EBV, CMV, herpesvirus 6, 7 or 8), hepadnavirus (HBV), papovavirus (HPV),poxvirus or retrovirus (HTLV, HIV).

The subject-matter of the present invention is also a compositioncharacterized in that it comprises at least one meganuclease, one or twoderived polynucleotide(s), preferably included in expression vector(s),as defined above.

In a preferred embodiment of said composition, it comprises a targetingDNA construct comprising the sequence which repairs the site of interestflanked by sequences sharing homologies with the targeted locus asdefined above. Preferably, said targeting DNA construct is eitherincluded in a recombinant vector or it is included in an expressionvector comprising the polynucleotide(s) encoding the meganuclease, asdefined in the present invention.

The subject-matter of the present invention is also products containingat least a meganuclease, or one or two expression vector(s) encodingsaid meganuclease, and a vector including a targeting construct, asdefined above, as a combined preparation for simultaneous, separate orsequential use in the prevention or the treatment of a genetic disease.

For purposes of therapy, the meganuclease and a pharmaceuticallyacceptable excipient are administered in a therapeutically effectiveamount. Such a combination is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of therecipient. In the present context, an agent is physiologicallysignificant if its presence results in a decrease in the severity of oneor more symptoms of the targeted disease and in a genome correction ofthe lesion or abnormality.

In one embodiment of the uses according to the present invention, themeganuclease is substantially non-immunogenic, i.e., engenders little orno adverse immunological response. A variety of methods for amelioratingor eliminating deleterious immunological reactions of this sort can beused in accordance with the invention. In a preferred embodiment, themeganuclease is substantially free of N-formyl methionine. Another wayto avoid unwanted immunological reactions is to conjugate meganucleasesto polyethylene glycol (“PEG”) or polypropylene glycol (“PPG”)(preferably of 500 to 20,000 daltons average molecular weight (MW)).Conjugation with PEG or PPG, as described by Davis et al. (U.S. Pat. No.4,179,337) for example, can provide non-immunogenic, physiologicallyactive, water soluble endonuclease conjugates with anti-viral activity.Similar methods also using a polyethylene-polypropylene glycol copolymerare described in Saifer et al. (U.S. Pat. No. 5,006,333).

The meganuclease can be used either as a polypeptide or as apolynucleotide construct/vector encoding said polypeptide. It isintroduced into cells, in vitro, ex vivo or in vivo, by any convenientmeans well-known to those in the art, which are appropriate for theparticular cell type, alone or in association with either at least anappropriate vehicle or carrier and/or with the targeting DNA. Once in acell, the meganuclease and if present, the vector comprising targetingDNA and/or nucleic acid encoding a meganuclease are imported ortranslocated by the cell from the cytoplasm to the site of action in thenucleus.

The meganuclease (polypeptide) may be advantageously associated with:liposomes, polyethyleneimine (PEI), and/or membrane translocatingpeptides (Bonetta, The Scientist, 2002, 16, 38; Ford et al., Gene Ther.,2001, 8, 1-4; Wadia and Dowdy, Curr. Opin. Biotechnol., 2002, 13,52-56); in the latter case, the sequence of the meganuclease fused withthe sequence of a membrane translocating peptide (fusion protein).

Vectors comprising targeting DNA and/or nucleic acid encoding ameganuclease can be introduced into a cell by a variety of methods(e.g., injection, direct uptake, projectile bombardment, liposomes,electroporation). Meganucleases can be stably or transiently expressedinto cells using expression vectors. Techniques of expression ineukaryotic cells are well known to those in the art. (See CurrentProtocols in Human Genetics: Chapter 12 “Vectors For Gene Therapy” &Chapter 13 “Delivery Systems for Gene Therapy”). Optionally, it may bepreferable to incorporate a nuclear localization signal into therecombinant protein to be sure that it is expressed within the nucleus.

The subject-matter of the present invention is also the use of at leastone meganuclease, as defined above, as a scaffold for making othermeganucleases. For example other rounds of mutagenesis andselection/screening can be performed on the variant, for the purpose ofmaking novel homing endonucleases.

The uses of the meganuclease and the methods of using said meganucleasesaccording to the present invention include also the use of thepolynucleotide(s), vector(s), cell, transgenic plant or non-humantransgenic mammal encoding said meganuclease, as defined above.

According to another advantageous embodiment of the uses and methodsaccording to the present invention, said meganuclease,polynucleotide(s), vector(s), cell, transgenic plant or non-humantransgenic mammal are associated with a targeting DNA construct asdefined above. Preferably, said vector encoding the monomer(s) of themeganuclease, comprises the targeting DNA construct, as defined above.

The polynucleotide fragments having the sequence of the targeting DNAconstruct or the sequence encoding the meganuclease variant orsingle-chain meganuclease derivative as defined in the presentinvention, may be prepared by any method known by the man skilled in theart. For example, they are amplified from a DNA template, by polymerasechain reaction with specific primers. Preferably the codons of the cDNAsencoding the meganuclease variant or single-chain meganucleasederivative are chosen to favour the expression of said proteins in thedesired expression system.

The recombinant vector comprising said polynucleotides may be obtainedand introduced in a host cell by the well-known recombinant DNA andgenetic engineering techniques.

The meganuclease variant or single-chain meganuclease derivative asdefined in the present the invention are produced by expressing thepolypeptide(s) as defined above; preferably said polypeptide(s) areexpressed or co-expressed (in the case of the variant only) in a hostcell or a transgenic animal/plant modified by one expression vector ortwo expression vectors (in the case of the variant only), underconditions suitable for the expression or co-expression of thepolypeptide(s), and the meganuclease variant or single-chainmeganuclease derivative is recovered from the host cell culture or fromthe transgenic animal/plant.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, CurrentProtocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley andson Inc, Library of Congress, USA); Molecular Cloning: A LaboratoryManual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, N.Y.:Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J.Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Harries & S. J. Higgins eds. 1984); TranscriptionAnd Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture OfAnimal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); ImmobilizedCells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide ToMolecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelsonand M. Simon, eds.-in-chief, Academic Press, Inc., New York),specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “GeneExpression Technology” (D. Goeddel, ed.); Gene Transfer Vectors ForMammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold SpringHarbor Laboratory); Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

In addition to the preceding features, the invention further comprisesother features which will emerge from the description which follows,which refers to examples illustrating the I-MsoI homing endonucleasevariants and their uses according to the invention, as well as to theappended drawings in which:

FIG. 1 represents the DNA targets. The C1234 wild-type I-CreI target andI-MsoI target are close derivatives: the two differences between the twotargets have been boxed in grey. They were first described as 24 bysequences but structural data indicate that only 22 by are relevant forprotein/DNA interaction. C1221 is the palindromic sequence derived fromthe left part of C1234. A 10NNN_P target is a derivative from C1221,where a degeneracy at positions ±10, ±9, ±8 has been introduced.

FIG. 2 represents the structure of the I-MsoI homing endonuclease incomplex with its DNA target according to Chevalier et al., J. Mol.Biol., 2003, 329, 253-269 (PDB code 1M5X).

FIG. 3 represents the area of the binding interface chosen forrandomization in this study. A. Molecular surface of I-MsoI bound to itsDNA target: base pairs at positions ±10, ±9, ±8 and protein residues 32,41 and 43 chosen for randomization are labeled in black. B. Zoom showingresidues 32, 41 and 43 in interaction with the nucleotides −10, −9 and−8 of the DNA target. Grey spheres are water molecules and dashed linesrepresent hydrogen bonds.

FIG. 4 represents the pCLS1055 reporter vector map. The reporter vectoris marked with TRP1 and URA3. The LacZ tandem repeats share 800 by ofhomology, and are separated by 1.3 kb of DNA. They are surrounded by ADHpromoter and terminator sequences. Target sites are cloned using theGateway protocol (Invitrogen), resulting in the replacement of the CmRand ccdB genes with the chosen target site.

FIG. 5 represents the pCLS0542 meganuclease expression vector map.pCLS0542 is a 2 micron-based replicative vector marked with a LEU2auxotrophic gene, and an inducible Gal10 promoter for driving theexpression of the I-MsoI variants.

FIG. 6 displays an example of primary screening of I-MsoI mutants fromthe Mlib1 library against 8 10NNN_P targets. Columns and rows arerespectively noted from 1 to 12 and from A to H. In each 9-dots yeastcluster, a Mlib1 mutant is screened against 8 different targets asexemplified by the experimental design. The bottom right dot is acluster internal control. Depending on the cluster, it is either anegative control (no meganuclease) either a positive control (weak orstrong versions of I-SceI, assayed on I-SceI target). H10, H11 and H12are also experiment controls.

FIG. 7 displays the hitmap of I-MsoI and I-MsoI variants against the 6410NNN_P targets. A. I-MsoI hitmap. B. Mlib1 library hitmap. Each novelendonuclease is profiled in yeast on a series of 64 palindromic targetsdescribed in FIG. 1, differing from the sequence shown in FIG. 1, atpositions ±8, ±9 and ±10. Each target sequence is named after the −10,−9, −8 triplet (10NNN). For example GGG corresponds to thecgggacgtcgtacgacgtcccg target (SEQ ID NO: 104). The number below eachcleaved target is the number of I-MsoI mutants with different sequencescleaving this target. For each target, the grey level is proportional tothe mean of cleavage intensity.

FIG. 8 displays represents the cleavage patterns of I-MsoI variantscleaving 31 DNA targets. For I-MsoI and each of the I-MsoI variants (SEQID NO: 6 to 99) obtained after screening and defined by the indicatedresidues, cleavage was monitored in yeast with the 64 targets describedin FIG. 7. Targets are designated by three letters, corresponding to thenucleotides at position −10, −9 and −8. For example GGG corresponds tothe cgggacgtcgtacgacgtcccg target (SEQ ID NO: 104; see FIG. 1). Valuescorrespond to the intensity of the cleavage, evaluated by an appropriatesoftware after scanning of the filter. The 13 targets which are notcleaved by I-MsoI are highlighted in grey with the correspondingvariants and their cleavage score.

FIG. 9 illustrates the correlation between given residues at positions32 and 41 of I-MsoI and bases at positions ±10; ±9 and ±8 (10NNN) of thetarget. The sum of all the intensities of cleavage from the matrix ofFIG. 8 are featured as a level of grey intensity, with a cumulatedintensity of 30 corresponding arbitrarily to black and 0 correspondingto white, for a mutant which has A, C, G, H, K, N, P, Q, R, S, T, W or Yat position 32 (left panel) or 41 (right panel) and tested with targetswhich have a, c, g or t at position −10, −9 or −8 (upper, medium andlower panel, respectively). The values are normalized to 100 by column.

EXAMPLE 1 Making of I-MsoI Derived Mutants Cleaving Degenerated 10NNN_PTargets

This example shows that I-MsoI mutants can cut DNA target sequencesderived from the C1221 target, a target efficiently cleaved by I-CreIand I-MsoI, and shown in FIG. 1. I-MsoI residues in direct or indirectinteraction with the DNA target nucleotides at position ±10; ±9 and ±8(10NNN) were pintpointed by a close examination of the structuredisplayed in FIG. 2. By direct interaction is meant a hydrogen bondbetween a protein residue and a base pair, an indirect interaction beinga water-mediated interaction between the protein and the DNA. Forexample, the residue R32 makes two hydrogen bonds with the guanine atposition −9 and contacts a water molecule, which itself interacts withthe adenine at position −10. Q41 and S43 are connected to the adenine atposition −8 via a water molecules network (FIG. 3). In order to isolatenew cleavage specificities for the I-MsoI protein, an I-MsoI mutantlibrary mutated at positions 32 and 41 (Mlib1) was built, transformed inthe yeast and screened against the 64 degenerated palindromic 10NNN_Ptargets (see FIG. 1) using the previously described screening assaybased on cleavage-induced recombination in yeast cells (InternationalPCT Application WO 2004/067736; Epinat et al., Nucleic Acids Res., 2003,31, 2952-2962; Chames et al., Nucleic Acids Res., 2005, 33, e178, andArnould et al., J. Mol. Biol., 2006, 355, 443-458). These assay resultsin a functional LacZ reporter gene which can be monitored by standardmethods. Such an approach has been already thoroughly described for theI-CreI protein (Smith et al., Nucleic Acids Res., 2006, 34, e149;International PCT Application WO 2007/049156).

1) Material and Methods a) Construction of the 64 Target Vectors

The targets were cloned as follows: oligonucleotides corresponding toeach of the 64 target sequences flanked by gateway cloning sequence wereordered from PROLIGO: 5′tggcatacaagtttcnnnacgtcgtacgacgtnnngacaatcgtctgtca 3′ (SEQ ID NO: 100).Double-stranded target DNA, generated by PCR amplification of the singlestranded oligonucleotide, was cloned using the Gateway protocol(INVITROGEN) into yeast reporter vector (pCLS1055, FIG. 4). Yeastreporter vector was transformed into S. cerevisiae strain FYBL2-7B (MATa, ura3Δ851, trp1Δ63, leu2Δ1, lys2Δ202) using a high efficiency LiActransformation protocol (Gietz and Woods, Methods Enzymol., 2002, 350,87-96).

b) Construction of the I-MsoI Mlib1 Mutant Library:

In order to generate I-MsoI derived coding sequences containingmutations at positions 32 and 41, separate overlapping PCR reactionswere carried out that amplify the 5′ end (aa positions 1-48) or the 3′end (positions 44-174) of the I-MsoI coding sequence (SEQ ID NO: 105).For the 3′ end, PCR amplification is carried out using a primer specificto the vector (pCLS0542, FIG. 5) (Gal10R 5′-acaaccttgattggagacttgacc-3′:SEQ ID NO: 101) and a primer specific to the I-MsoI coding sequence foramino acids 44-56 (MlibF1 5′-ctagcaatttatttatacaaagaaaagataaatttcc-3′:SEQ ID NO: 102). For the 5′ end, PCR amplification is carried out usinga primer specific to the vector pCLS0542 (Gal10F5′-gcaactttagtgctgacacatacagg-3′: SEQ ID NO: 103) and a primer specificto the I-MsoI coding sequence for amino acids 29-48 (Mlib1R5′-aaaagaaattgctagactcacmbnatatttaatgtctttgtaatcaggmbnaggaataag-3′(SEQID NO: 106). The mbn code in the oligonucleotide resulting in a NVKcodon at position 32 and 41 allows the degeneracy at these positionsamong a group of 15 possible amino acids (S, P, T, A, Y, H, Q, N, K, D,E, C, W, R and G). Then, 25 ng of each of the two overlapping PCRfragments and 75 ng of vector DNA (pCLS0542) linearized by digestionwith NcoI and EagI were used to transform the yeast Saccharomycescerevisiae strain FYC₂₋₆A (MATα, trp1Δ63, leu2Δ1, his3Δ200) using a highefficiency LiAc transformation protocol (Gietz and Woods, MethodsEnzymol., 2002, 350, 87-96). An intact coding sequence containing bothgroups of mutations is generated by in vivo homologous recombination inyeast. The Mlib1 nucleic diversity is 24²=576, so after transformation,1116 clones, around two times the library diversity, were picked.

c) Mating of Meganuclease Expressing Clones and Screening in Yeast

Mating was performed using a colony gridder (QpixII, GENETIX). Mutantswere gridded on nylon filters covering YPD plates, using a low griddingdensity (about 4 spots/cm²). A second gridding process was performed onthe same filters to spot a second layer consisting of differentreporter-harboring yeast strains for each target. Membranes were placedon solid agar YPD rich medium, and incubated at 30° C. for one night, toallow mating. Next, filters were transferred to synthetic medium,lacking leucine and tryptophan, with galactose (1%) as a carbon source,and incubated for five days at 37° C., to select for diploids carryingthe expression and target vectors. After 5 days, filters were placed onsolid agarose medium with 0.02% X-Gal in 0.5 M sodium phosphate buffer,pH 7.0, 0.1% SDS, 6% dimethyl formamide (DMF), 7 mM β-mercaptoethanol,1% agarose, and incubated at 37° C., to monitor β-galactosidaseactivity. Results were analyzed by scanning and quantification wasperformed using appropriate software.

d) Sequencing of Mutants

To recover the mutant expressing plasmids, yeast DNA was extracted usingstandard protocols and used to transform E. coli. Sequencing of mutantORF was then performed on the plasmids by MILLEGEN SA. Alternatively,ORFs were amplified from yeast DNA by PCR (Akada et al., Biotechniques,2000, 28, 668-670), and sequence was performed directly on PCR productby MILLEGEN SA.

2) Results

Using the yeast screening assay that has been described above, the 1116clones that constitute the I-MsoI Mlib1 library were screened againstthe 64 10NNN_P targets. The screen gave 246 positive clones able tocleave at least one 10NNN_P target (FIG. 6), resulting after sequencingin 94 unique meganucleases. The I-MsoI protein is able to cleave 18 outof the 64 10NNN_P targets (FIG. 7A). The Mlib1 hitmap displayed in FIG.7B shows that by introducing mutations at positions 32 and 41 in theI-MsoI coding sequence, 13 new additional 10NNN_P targets are now beingcleaved by I-MsoI derived mutants. The cleavage pattern of the variantsis described in FIG. 8. This screening approach has therefore allowed towiden the I-MsoI cleavage spectrum of 10NNN_P targets and to isolate newcleavage specificities.

EXAMPLE 2 Analysis of Correlation Between Given Residues at Positions 32and 41 of I-MsoI and Bases at Positions ±10; ±9 and ±8 (10NNN) of theTarget

To identify potential correlation between specific residues at positions32 and 41 of I-MsoI and bases at positions ±10; ±9 and ±8 (10NNN) of thetarget, a statistical analysis of the positives was conducted.

1) Materials and Methods

From the initial (mutant,target) matrix, and for each pair (p, q) ofmutated amino-acid position ‘p’ on the protein and nucleic acid position‘q’ on the target, a matrix of cumulated intensities was computed fromthe data from FIG. 8. This matrix of cumulated intensities has a numberof columns equal to the number of distinct amino-acids occurring at p onour set of mutants and 4 rows (one for each nucleotide). The value ofthis matrix for amino-acid value ‘A’ and nucleotide ‘N’ is the sum ofall the intensities of the initial matrix for mutants which have an A atposition p and tested with targets which have an N at position q. OnFIG. 9, these values are featured as a level of grey intensity, with acumulated intensity of 30 corresponding arbitrarily to black and 0corresponding to white. Then, this matrix was normalized to 100 bycolumn (sum of all the cells for each column equal to 100). An imagecorresponding to each matrix was drawn, with the normalized valuewritten in each cell.

2) Results

Results are summarized in the six panels from FIG. 9. Only dark cellsare significant since the brighter cells represent only low levels ofcuttings.

Potentially significant correlations between amino-acids and nucleotidesshould correspond to amino-acids with highly differing percentagereparation along the four nucleotides while cells are dark enough toensure significance. R32 (found in the wild type protein as well as inseveral mutants) and bases G, G and A at positions ±10; ±9 and ±8 of thetargets are often associated, revealing the overepresentation of anearly wild type profile (note that the sequence of the wild type targetI-MsoI is AGA in −10, −9 and −8, top strand, and GAA in 8, 9, 10, bottomstrand). No other significant association could be inferred from thesample of positives, showing an absence of correlation betweenindividual residues at position 32 and 41 and bases ±10; ±9 and ±8 ofthe targets. Thus, one can hypothesize that specificity for eachspecific base is largely influenced by more than one protein residue.

1. An I-MsoI variant which has at least one substitution at least one ofpositions 31, 32, 33, 35, 41, and 43 of I-MsoI, wherein the at least onesubstitution is selected from the group consisting of: replacement ofP31 or P33 with S, T, A, Y, H, Q, N, K, D, E, C, W, R or G, replacementof R32 with Q, A, H, S, G, D, W, P, T, C, or N. replacement of Y35 withS, P, T, A, H, Q, N, D, E, C, W, or G, replacement of Q41 with N, G, Y,T, S, P, C, H, A or W, replacement of Y35 with S, T, A, H, Q, N, K, D,E, C, W, R or G, and replacement of S43 with P, T, A, Y, H, N, D, C, W,or G, wherein said variant is able to cleave a panel of mutant I-MsoIsites having variation at positions ±8 to 10 that is different fromsites cleaved by I-MsoI.
 2. The variant according to claim 1, comprisingat least one additional substitution at a position of I-MsoI thatimproves at least one of binding and cleavage activities towards a DNAtarget, wherein the at least one additional substitution is selectedfrom the group consisting of. T3, K4, T6, L7, K36, D37, K39, Y40, V42,F48, F55, Y82, T88, 193, L97, N109, I134, A145, T151 and A163.
 3. Thevariant according to claim 2, wherein said substitution is selected fromthe group consisting of. T3A, K4M, T6A, L7S, K36N, K361, D3N7, K39N,K39R, K39T, Y40S, V42M, F48Y, F55V, F55I, Y82H, T88A, I93M, L97S, N109S,I134V, I134M, A145V, T151A and A163V.
 4. The variant according to claim1, which is able to cleave at least one target that is not cleaved byI-MsoI, said variant comprising substitutions selected from the groupconsisting of. R32K and Q41N; Q41T; R32S and Q41S; R32A and Q41R; R32Wand Q41N; R32S and Q41R; R32Q and Q41R; Q41Y; Q41N; Q41C; R32T and Q41R;Q41H; R32W and Q41T; Q41S; Q41G; R32E and Q41T; R32Q and Q41A; R32G andQ41Y; Q41P; R32P and Q41T; Q41A; T3A, R32Q and Q41P; Q41N and T88A; R32Sand Q41N; R32Q, Q41P and F48Y; R32S, K39N and Q41S; R32D, Q41K and L97S;R32H, Q41K and A145V; P33S and Q41C; Y35F and Q41K; R32C, K39T and Q41K;R32A and Q41P; R32T, Y40S and Q41S; R32G and Q41R; R32H and Q41P; R32E,K36E and Q41T; R32P and Q41P.
 5. The variant according to claim 1, whichcleaves less targets than I-MsoI, said variant comprising substitutionsselected from the group consisting of. R32Q and Q41G; R32A and Q41Y;R32H and Q41R; R32D and Q41P; R32D and Q41R; R32Q and Q41N; R32P andQ41R; R32K and Q41Y; R32K and Q41T; R32K and Q41H; R32K, Q41G and V42M;R32S and Q41Y; R32H and Q41G; R32H and Q41H; R32Q and Q41S; R32S andQ41K; R32A and Q41S; R32H and Q41S; R32C and Q41H; R32H and Q41N; R32Cand Q41T; R32S and Q41H; R32T and Q41K; R32A and Q41H; R32G and Q41K;R32S and Q41P; R32H and Q41T; R32Q and Q41H; R32Q and Q41T; R32K andQ41R; R32E and Q41W; R32K and Q41S; R32N and Q41N; R32H and Q41C; R32Sand Q41A; Q41K and F55I; T6A, Q41K and 193M; R32E, Q41T and N109S; R32Gand Q41W; K4M, R32T and Q41R; Y35S and D37N; R32H and Q41A; K39R andQ41S; L7S, R32K and Q41H; K36N and Q41N; P33L and Q41P; R32T, Q41R andT151A; Q41Y and A163V; R32S, Q41H and I134V; Q41T and Y82H; R32H, D37Nand Q41T; Q41N and P43N; R32K, Q41S and I134M; R32A, Q41K and F55V; Q41Sand F48Y.
 6. The variant according to claim 1, wherein the variant is anhomodimer.
 7. The variant according to claim 1, wherein the variant isan heterodimer comprising two different variants according to claim 1.8. A single-chain chimeric meganuclease obtained from the variantaccording to claim 1, comprising two monomers, two core domains or acombination of one monomer and one core domain from said variant.
 9. Apolynucleotide fragment encoding at least one monomer of the variant ofclaim
 1. 10. An expression vector comprising at least one polynucleotidefragment of claim 9, wherein the at least one polynucleotide fragment isoperatively linked to regulatory sequences allowing production of saidvariant.
 11. The vector of claim 10, comprising targeting DNA constructcomprising sequences sharing homologies with a region surrounding agenomic DNA target sequence that is cleaved by said meganuclease variantor single-chain meganuclease.
 12. The vector of claim 11, wherein saidtargeting DNA construct comprises. a) sequences sharing homologies withthe region surrounding the genomic DNA target sequence that is cleavedby said meganuclease variant or single-chain meganuclease, and b)sequences to be introduced flanked by a).
 13. A host cell comprising atleast one polynucleotide fragment according to claim
 9. 14. A non-humantransgenic animal comprising one polynucleotide fragment according toclaim
 9. 15. A transgenic plant comprising at least one polynucleotidefragment according to claim
 9. 16. A pharmaceutical compositioncomprising at least a variant of claim
 1. 17. The composition of claim16, which comprises a targeting DNA construct comprising a sequencewhich repairs a genomic site of interest flanked by sequences sharinghomologies with a targeted locus.
 18. (canceled)
 19. (canceled)
 20. Amethod of treating a disease caused by an infectious agent that presentsa DNA intermediate, in an individual in need thereof, comprisingadministering the variant of claim 1 to the individual.
 21. A method forinhibiting an infectious agent that presents a DNA intermediate, in abiological derived product intended for biological uses or fordisinfecting an object, the method comprising incorporating the variantof claim 1 into the product.
 22. The method according to claim 20,wherein said infectious agent is a virus.
 23. The method according toclaim 18, wherein said variant is associated with a targeting DNAconstruct.
 24. A scaffold for engineering a meganuclease, comprising thevariant according to claim
 1. 25. A method for genetic engineering,comprising generating a double-strand break in a site of interestcomprising at least one recognition and cleavage site of a variantaccording to claim 1, by contacting said recognition and cleavage sitewith said variant, wherein said double-strand break thereby induces aDNA recombination event, a DNA loss or cell death.
 26. A method forpreventing, improving or curing a genetic disease in an individual inneed thereof, comprising administering the variant of claim 1 to theindividual.